Hemolysin Co-regulated Family Proteins Hcp1 and Hcp2 Contribute to Edwardsiella ictaluri Pathogenesis

Abstract

Edwardsiella ictaluri is a Gram-negative facultative intracellular pathogen causing enteric septicemia of catfish (ESC), a devastating disease resulting in significant economic losses in the U.S. catfish industry. Bacterial secretion systems are involved in many bacteria's virulence, and Type VI Secretion System (T6SS) is a critical apparatus utilized by several pathogenic Gram-negative bacteria. E. ictaluri strain 93–146 genome has a complete T6SS operon with 16 genes, but the roles of these genes are still not explored. In this research, we aimed to understand the roles of two hemolysin co-regulated family proteins, Hcp1 (EvpC) and Hcp2. To achieve this goal, single and double E. ictaluri mutants (EiΔevpC, EiΔhcp2, and EiΔevpCΔhcp2) were generated and characterized. Catfish peritoneal macrophages were able to kill EiΔhcp2 better than EiΔevpC, EiΔevpCΔhcp2, and E. ictaluri wild-type (EiWT). The attachment of EiΔhcp2 and EiΔevpCΔhcp2 to ovary cells significantly decreased compared to EiWT whereas the cell invasion rates of these mutants were the same as that of EiWT. Mutants exposed to normal catfish serum in vitro showed serum resistance. The fish challenges demonstrated that EiΔevpC and EiΔevpCΔhcp2 were attenuated completely and provided excellent protection against EiWT infection in catfish fingerlings. Interestingly, EiΔhcp2 caused higher mortality than that of EiWT in catfish fingerlings, and severe clinical signs were observed. Although fry were more susceptible to vaccination with EiΔevpC and EiΔevpCΔhcp2, their attenuation and protection were significantly higher compared to EiWT and sham groups, respectively. Taken together, our data indicated that evpC (hcp1) is involved in E. ictaluri virulence in catfish while hcp2 is involved in adhesion to epithelial cells and survival inside catfish macrophages.

Edwardsiella ictaluri: A systemic review and future perspectives on disease management

“…105 Consequently, intracellular survival of E. ictaluri is enabled by the upregulated expression of T3SS by the two-component regulatory proteins EsrA and EsrB 106 and the activity of the type VI secretion system (T6SS) effector, Hcp2. 101 Also, using urea that would have been produced by arginase enzyme from the host cell, the E. ictaluri acid-activated urease produces ammonia, which neutralizes the ECV acidic environment to a pH level (>pH 5.0). This creates an environment conducive for E. ictaluri replication and translocation of T3SS effectors (EseGHIJKLMNO) directly into the host cytoplasm.…”

“…The ECV is acidified immediately by host cell vacuolar ATPases 105 . Consequently, intracellular survival of E. ictaluri is enabled by the upregulated expression of T3SS by the two‐component regulatory proteins EsrA and EsrB 106 and the activity of the type VI secretion system (T6SS) effector, Hcp2 101 . Also, using urea that would have been produced by arginase enzyme from the host cell, the E. ictaluri acid‐activated urease produces ammonia, which neutralizes the ECV acidic environment to a pH level (>pH 5.0).…”

“…The molecular mechanisms of E. ictaluri pathogenesis were described in channel catfish and zebrafish using epithelial cells, phagocytic cells and macrophages, and a graphical illustration is shown in Figure 1b. Pathogen attachment is facilitated by the recognition of E. ictaluri by host cell receptors, for example Toll‐like receptor 5 (TLR5) and (NOD)‐like receptor subfamily C (NLRC) 100 and the help of E. ictaluri proteins Hcp2, 101 EseI and EseH 102 . For invasion, the plasmid‐encoded protein, EseI plays a role, 102 and E. ictaluri enters into the target cells using Ca 2+ ‐dependent receptor‐mediated endocytosis and macropinocytosis 103,104 .…”

“…Conducive pH is then maintained by the prevention of phagosomal/lysosomal fusion, nutrients for bacterial growth and ECV enrichment are supplied by the Golgi, and programmed cell death is suppressed 109 . Lysosomal acid hydrolases and reactive oxygen species production is downregulated by the T6SS effector, EvpP, indicating exploitation of the endosomal machinery, thereby enabling intra‐phagosome survival 100,101 . Lastly, inflammatory and immune responses are modulated with the putative aid of the EseN protein, for disease progression, and then, genes responsible for endocrine and growth are downregulated which may contribute to faltering growth 100 .…”

Hemolysin Co-Regulatory Protein 1 Enhances the Virulence of Clinically Isolated Escherichia coli in KM Mice by Increasing Inflammation and Inducing Pyroptosis

“…Hemolysin-coregulated protein 1 (Hcp1) is a member of the Hcp protein family found in Escherichia coli ( E. coli ), which is unique to T6SS and makes up the bulk of its extracellular structure [ 7 , 8 ]. Hcp1 contributes to the virulence of Edwardsiella ictaluri in catfish [ 9 ]. In Burkholderia pseudomallei , Hcp1 stimulates strong IFN-γ-secreting T-cell responses in patients with acute melioidosis and correlates with patient survival [ 10 ].…”